ABSTRACT A single human genome gives rise to hundreds of differentiated cell types that must maintain their distinct identities for proper development and to prevent disease, most notably cancer. A complex regulatory layer has evolved to maintain the transcriptional profiles of differentiated cells, which often involves pathways that modify chromatin chemically, by adding or removing small molecular tags to histone proteins and DNA, or physically, by compacting it into heterochromatin or remodeling it to make it more accessible. Proteins and complexes of the Polycomb group modify chromatin chemically and physically. The Polycomb repressive complex 2 (PRC2) interprets the transcriptional and epigenetic state of the nucleus and try- methylates histone H3 at lysine 27 (H3K27me3), a chemical mark that imposes self-propagating, chromatin- based silencing. The Polycomb repressive complex 1 (PRC1) integrates a variety of signals, including H3K27me3 to enforce transcriptional repression. The full extent of the molecular signals that converge on PRC1 and the mechanism by which PRC1 imposes transcriptional repression remain poorly understood. The central hypothesis tested in this proposal is that SCMH1, a Polycomb protein of the malignant brain tumor (MBT) family, plays a key role in chromatin compaction and transcriptional repression by PRC1 and that its function is regulated by interactions with noncoding RNAs. Using mouse embryonic stem cells (ESCs) as a model system, we will test this hypothesis with two specific aims. In aim 1, we will determine the cellular and biochemical function of SCMH1 in the context of PRC1-mediated gene repression. We will generate ESC lines carrying Scmh1 loss-of-function (KO) alleles using CRISPR/Cas9 and analyze their effects on transcription and chromatin structure in undifferentiated and differentiated ESCs. ChIP-seq for SCMH1 and PRC1 core subunits in control and Scmh1 KO cells will reveal the relationship at genome-wide level. Rescue of KO cells with different SCMH1 mutants will offer insight on the requirements and molecular mechanism of PRC1 regulation by SCMH1. In vitro experiments with reconstituted PRC1 complex and recombinant SCMH1 will further dissect the biochemical function of SCMH1. In aim 2, we will utilize established and novel techniques to map the region of SCMH1 that interacts with RNA and to identify RNAs bound to SCMH1. We will design and test mutants of SCMH1 to impair its ability to interact with RNA while retaining all other biochemical function and we will study to what extent those mutants rescue the function of Scmh1 KO described above both using in vivo functional genomics readouts as well as in vitro biochemical assays. Our studies will offer much needed insight on the function and regulation of PRC1, a key gatekeeper of cellular identity, and on the role of noncoding RNAs in Polycomb-mediated epigenetic repression.